This invention relates to a DNA fragment having a promoter activity in plants. More particularly, this invention relates to a novel DNA fragment having a promoter activity derived from a gene encoding metallothionein and a vector containing the same and a plant cell, a plant and a seed transformed thereby.
Improvement of plants by using a genetic engineering method has become practical recently. Particularly, a mechanism of tumor formation in plant by Ti plasmid of Agrobacterium tumefaciens which is a soil microorganism is solved in molecular level, and taking this opportunity, since a transformation system using a Ti plasmid vector has been established, research on plants in molecular level has been making a significant progress. In these days, the transformation system using the Ti plasmid vector can be applied to a main agricultural products including rice, soy been, etc., as well as to the model plants such as tobacco and Arabidopsis thaliana, etc.
It has been generally known that a 5xe2x80x2 upstream region of a structural gene called promoter is involved in a transcription of a gene. The promoter is a DNA sequence located in an upstream region of a structural gene, containing a signal (TATA region) for an RNA polymerase to start transcription, thereby enabling a following protein synthesis. Therefore, the promoter is an important and essential gene to produce a genetically recombinant plant.
In a 5xe2x80x2 upstream of the TATA region, there exists a specific nucleotide sequence called cis element, and this region interacts with a DNA binding protein (trans element) to determine a strength of promoter activity and control of the transcription.
For example, a promoter of Arabidopsis thaliana gene (rd29A) whose expression is induced by drought [Koizumi et al., Gene 129:175-182 (1993)] was isolated, and after that, from a research in which a promoter region is mutated by deletion or a research in which various kinds of DNA fragments are linked, it has been reported that the cis element of rd29A gene controlling an induction by drought is a 9 base sequence comprising TACCGACAT [Yamaguchi-Shinozaki et al., J. Plant Res. 108:127-136 (1995)], and it has been elucidated that the above mentioned cis element comprising 9 bases is essential for serving as a promoter induced by drought.
Currently, as a promoter for a gene transduction, 35S promoter of a cauliflower mosaic virus [Guilley et al., Cell 30:763-773 (1982)] has been frequently used. From analyses using tobacco [Morell et al., Nature 315:200-204 (1985)] on petunia [Sander, Nucl. Acid Res. 15:1543-1558 (1987)], 35S promoter has been shown to have an activity 30 times or more as strong as that of nopaline synthetase promoter. As shown above, since the promoter activity of 35S promoter is strong in dycotyledons, this promoter is widely used in order to transfer a structural gene and have it highly expressed in dycotyledons.
However, 35S promoter shows only relatively low promoter activity in a rice family plant which is an important monocotyledon in agriculture [Hauptmann et al., Plant Cell Rep. 6:265-270 (1987)].
On the contrary, a promoter derived from alcohol dehydrogenase (Adh) of corns merely gives an extremely low expression in protoplast of Nicotiana plumbaginifolia which is a dicotyledon [Ellis et al., EMBO J. 6:11-16 (1987)].
In order to produce practically useful novel plant strain in a wide range of plants by recombinant DNA technology in the future, it is thought to be an important technology, how to carry out efficiently an expression of a structural gene to be transferred or how to control an expression. However, with the 35S promoter or the Adh promoter, it is not easy to control a tissue-specific expression, or to control an expression by chemical substances.
Therefore, a promoter having a promoter activity not only in monocotyledons but also in dicotyledons, and being able to control a tissue specific and part specific expression has been sought.
Incidentally, metallothionein (Mt) has been known as a protein with a low molecular weight, playing an important role in metabolism of metals in vivo in animals, microorganisms and plants [Yu et al., Gene 206:29-35 (1998)].
Mt was initially isolated from the kidneys of horses and research on its structure and its function has made progress by using organisms such as cyanobacteria or fungi as a sample [Yu et al., Plant Biotechnology 15:167-172 (1998)]. When Mt originated from Chinese hamster is expressed in a metallo-sensitive yeast, the yeast becomes resistant to cadmium. This suggests Mt takes a part in detoxification of metals.
Regarding rice Mt gene, cDNA has been isolated from YAMAHOUSHI, and a promoter region has been isolated from Sasanishiki, whose base sequences have been reported [Japanese Provisional Patent Publication No. 10-248570].
The present invention has been aimed to obtain and provide a novel DNA fragment having a promoter activity which makes a linked exogenous structural gene express in the plant or plant cells, and enabling an induction of expression or control of a tissue-specific and part-specific expression by substances.
The present inventors have made intensive studies to solve the above-mentioned problems and they have isolated a promoter region of Mt gene from rice plant NNipponbare. As a result of comparison of nucleotide sequences with a promoter region isolated from Sasanishiki, the both sequences are totally different and a novel promoter has been identified. Further, they have found that this promoter has a promoter activity in a different plant strain, regardless of monocotyledons or dicotyledons, and that it enables an expression of an exogenous gene and control of the expression, and thus, the present invention has been completed.
The present invention comprises promoters shown below, which is derived from a gene encoding a rice metallothionein. That is, it is a DNA fragment having a promoter activity comprising a whole or a part of a base sequence shown by SEQ ID NO:1 wherein one or two or more bases maybe deleted, inserted or substituted provided that the sequence has a function in plants or in plant cells to control expression of a structural gene which can be expressed in plants, for example, a DNA of about 2.7 Kbp shown by SEQ ID NO:2.
Further, the present invention is an expression vector into which a DNA fragment shown by SEQ ID NO:1 or 2 is transferred.
In addition, the present invention relates to an expression vector wherein the DNA sequence shown by SEQ ID NO:1 or 2 is transferred to an exogenous structural gene.
Yet further, the present invention relates to a transformed plant cell obtainable by introducing the above-mentioned vector into a host plant cell, a transformed plant regenerated from the plant cells and a seed obtainable from the plant.
In the description below, unless otherwise specified, recombinant DNA technology used as a conventional method can be carried out using techniques described in the following reference: xe2x80x9cMolecular Cloningxe2x80x9d (Fristch et al., Cold Spring Harbour Press (1989)).
The DNA fragment having a promoter activity of the present invention can be isolated from a plant genomic library by a plaque hybridization, etc. using cDNA of rice metallothionein as a probe.
Genomic library can be obtained by extracting genomic DNA from plant such as a rice plant, partially digesting the isolated genomic DNA by an appropriate restriction enzyme such as EcoRI, fractionating longer DNA fragments, for example, DNA fragments of 9 to 23 kb by sucrose density-gradient centrifugation or by agarose gel electrophoresis, incorporating this into an appropriate vector such as xcex phage and packaging, followed by infecting Escherichia coli such as XL-1 Blue strain with this recombinant phage, and culturing them on a flat culture medium such as an LB plate (1% Bactotryptone, 0.5% yeast-extract, 1% NaCl and 1.3% agarose). As a vector, a plasmid may be used, however, it is preferable to use xcex phage or cosmid vector in that longer DNA fragments can be effectively inserted.
Membrane such as nylon membrane is placed on the above-mentioned flat culture medium, and this membrane is immersed in a denaturing agent, followed by immersing in a neutralizing solution and washing with a washing solution, so that the recombinant DNAs on the flat culture medium become single stranded and are adsorbed onto the membrane.
As a probe, for example, an oligo DNA synthesized according to a part of Mt structural gene or a DNA amplified by the PCR method using Mt cDNA is labeled with [xcex1-32P]dCTP, DIG (Digoxigenin), biotin, etc. and used.
Hybridization can be carried out using the membrane to which the above-mentioned single stranded DNAs are adsorbed and the labeled probe.
The membrane thus hybridized with the labeled probe is applied to autoradiography to detect clones strongly hybridized with the probes, and they can be isolated as positive clones.
DNA of the isolated positive clone is treated with an appropriate restriction enzyme, such as Eco RI, carrying out subcloning with an appropriate cloning vector such as pBluescript II and pUC type vector, and then, its nucleotide sequence can be determined by the Maxam-Gilbert method or by dideoxy method, etc. Commercially available kits, and an automated sequencer that automatically determines a sequence, etc. may be used.
Base sequence of the obtained clone is determined as mentioned above and the 5xe2x80x2 non-translated region and its upstream promoter region of the Mt gene are found out.
To a downstream of the thus obtained promoter region, a structural gene and a terminator gene are linked, and by inserting them into an expression vector, it is possible to construct an expression vector for gene transfer.
As an expression vector, there may be mentioned pUC type vector (for example, pUC118, pUC119), pBR type vector (for example, pBR322), pBItype vector (for example, pBI101, pBI112 and pBI221), pGA type vector (pGA492 and pGAH), etc. In addition to these, virus vectors, etc. may be listed.
As a terminator gene to be linked, there may be mentioned 35S terminator gene, Nos terminator gene, etc.
As a structural gene to be linked, there may be mentioned, for example, a reporter gene, a gene of an insecticidal protein, a herbicide-resistant gene, a gene expressing antibacterial activity, blight resistant gene, a gene which induces bloom of plants, a gene involving morphogenesis, a gene involving resistance to an environmental stress, a gene involving in increasing an yield, a gene with an antiseptic effect on crops, etc.
As a reporter gene, there may be mentioned xcex2-glu-curonidase (GUS) gene, luciferase gene, chloramphenicol acetyltransferase (CAT) gene, etc. As a gene of an insecticidal protein, there may be mentioned a gene of a crystalline protein of Bacillus thuringiensis, a protease inhibitor gene, etc., as a herbicide-resistant gene, a gliphosate-resistant gene, a glufosinate-resistant gene, a sulfonylurea type herbicide resistant gene, etc., as a gene expressing antibacterial activity, chitinase gene, glucanase gene, lysozyme gene, cecropin gene, etc., as a gene which induces bloom of plants, a gene involving formation of florigen, etc., as a gene involving morphogenesis, chalcone synthase gene, phenylalanine ammonia-lyase, RolC gene, etc., as a gene involving resistance to an environmental stress, glutamine synthase gene, glycine betaine gene, Mt gene, etc., as a gene involving in increasing a yield, a modified gene of a seed storage protein, sucrose-phosphate synthase gene, etc., and as a gene with an antiseptic effect on crops, an antisense gene of ethylene synthase gene, etc.
As a method for introducing an expression vector in which a structural gene such as a reporter gene and a terminator gene are linked to a downstream of a promoter, there maybe mentioned an indirect introducing method or a direct introducing method.
As the indirect introducing method, for example, a method using Agrobacterium is exemplified.
As the direct introducing method, there may be exemplified, for example, an electroporation method, a particle gun method, a polyethylene glycol method, a microinjection method, a silicon carbide method, etc.
All kinds of plants are included as a plant to which a gene is transferred, and, for example, there may be mentioned the rice family, the palm family, the lily family, the orchid family, the taro family, etc., for monocotyledon plants.
Examples of the rice family may include a rice plant, wheat, barley, ryewheat, brushwood, a ditchreed, asugarcane, corn, foxtail millet, a barnyard grass, etc. Examples of lily family may include a Welsh onion, a lily, a tulip, etc.
For dicotyledon plants, examples may include the beech family, the cactus family, the camellia family, the mallow family, the gourd family, the rape family, the rose family, the pulse family, the mulberry family, Euphorbia family, the grape family, the tangerine family, the drop wort family, the eggplant family, the perilla family, the crysanthemum family, the primrose family, the fringed pink family, etc. As the camellia family, tea, etc. are mentioned. As the mallow family, cotton, etc. are mentioned. As the gourd family, cucumber, melon, pumpkin, etc. are mentioned. As the rape family, rape, Arabidopsis thaliana, Japanese radish, horseradish, cabbage, etc. are mentioned. As the rose family, Japanese apricot, peach, apple, pears, rose, etc. are mentioned. As the pulse family, soybean, adzuki bean, garden pea, broad bean, peanut, etc. are mentioned. As the mulberry family, hop, etc. are mentioned. As the eggplant family, tobacco, eggplant, potato, tomato, etc. are mentioned. As the crysanthemum family, crysanthemum, garden crysanthemum, sunflower, lettuce, etc. are mentioned. As the primrose family, primrose, cyclamen, etc. are mentioned. As the fringed pink family, carnation, etc. are mentioned.
Further, plants which belong to a gymnosperm, such as Japanese cedar family, pine tree family, white cedar family, etc. are also included.
A plant which contains the transferred promoter and the structural gene can be obtained by inserting a gene having a resistance to chemicals such as kanamycin, hygromycin, etc. to a gene transferred vector, or by simultaneously introducing a vector having a chemical resistant gene to a plant, and then, selecting with a chemical such as kanamycin or hygromycin, etc. Further, by analyzing the gene transferred into the plant, such as a PCR method or a southern hybridization method, etc., or by analyzing a translated product of the transferred structural gene, for example, by analyzing a desired protein contained in an extract of the plant leaves, etc. in terms of enzyme activity or by a Western blotting method, etc., thereby confirming and selecting plants to which the desired gene is transferred.
It is possible to grow the thus obtained transformed plant to collect its seeds, and to differentiate and regenerate a plant using a tissue of the transformed plant, such as a leaf or a root.
Since Mt gene is localized in the internode, it is possible to control site of expression of the transferred gene in the transformed plant into which it is transferred, by linking a structural gene to a downstream of the promoter region.
Analysis on site-specific expression of the structural gene which is linked and transferred into the promoter is carried out by analyzing localization of mRNA or a translated product of the structural gene in the plant.
For example, when a GUS gene is used as a structural gene, an existing site of GUS in the transformed plant can be confirmed by a histochemical method.
That is, localized site of GUS can be confirmed by observing tissue by a microscope for a blue color of indigotin, that is a hydrolysis product of 5-bromo-4-chloro-3-indolyl-xcex2-D-glucuronic acid by GUS.